The phosphorylated and dephosphorylated forms of myosins IA and IB bind identically to F-actin. The kinetics of ATPase activity as a function of actin concentration shows three phases: activation, inactivation and reactivation, that are interpretable in terms of a mechanism suggesting the presence of two actin-binding sites on each myosin molecule. A protein phosphatase that removes all three phosphate groups from the heavy chains of myosin II (and activates its actomyosin ATPase activity) has been purified to homogeneity and characterized. The 9,000-dalton phosphorylated peptide segment of the heavy chain of myosin II has been partially sequenced. It has been shown that both phosphorylated and dephosphorylated myosin II are filamentous under assay conditions in vitro, that dephosphorylated myosin II are filamentous under assay conditions in vitro, that dephosphorylated myosin II forms larger filaments under equivalent conditions and that copolymers of phosphorylated and dephosphorylated myosin II are more similar in size to the phosphorylated homopolymer. The presence of the phosphorylated molecules in the co-polymers inactivates the actin-activated ATPase activity of the dephosphorylated molecules. Regulation by phosphorylation apparently involves an effect on the conformation of the filament as a whole rather than an intramolecular or direct intermolecular effect.